Neurosteroids [allopregnanolone (ALLO), progesterone (PROG), 5alpha- dehydroprogesterone (5alpha-DHP)] are pharmacologically potent modulators of neuronal activity either by regulating GABA and glutamate receptor functions, or by controlling specific protein gene expression. Within the brain, glial cells contain on the inner mitochondrial membrane an enzyme (P450acc) needed to synthesize pregnenolone (PREG) from cholesterol. Glial cells also contain a receptor on the outer layer of mitochondria called MDR [for mitochondrial diazepam binding inhibitor (DBI)] which favors the intramitochondrial transfer of cholesterol to the p450acc enzyme. In spite of the high level of interest triggered by these discoveries, work previous to this application did not establish whether significant amounts of pharmacologically active neurosteroids are produced in the brain. Because chemical and radioimmuological detection methods are not available for accurate measurement of small quantities of neurosteroids and their processing products, progress in establishing the physiological relevance of neurosteroids has been difficult. The first Aim of our research has been to develop a specific and sensitive method combining high performance liquid chromatography (HPLC) and gas chromatography-mass fragmentography (GC-MF) which will allow the detection of a large number of neurosteroids, their precursors and metabolic products in small tissue samples and biological fluids. Using the new HPLC/GC-MF method and administering to adrenalectomized/castrated (ADX/CX) rats appropriate blockers of the 5- alpha-reductase (SKF105,111) and of the 3alpha hydroxysteroid oxidoreductase (3alpha-HSOR) (indomethacin) we have demonstrated that ALLO is synthesized nonuniformly in the brain by the coordinated action of 5alpha-reductase and 3alpha-HSOR with the highest concentration (approximately 10 pmol/mg prot) in olfactory bulb. The brain content of ALLO does not depend on peripheral organs because; a) peripheral tissues of ADX/CX rats to not synthesize ALLO and b) ADX/CX virtually eliminate ALLO from plasma but reduce only marginally the content of ALLO in brain. Moreover, ALLO is found in significant concentrations in brain microdialysates. This data strongly suggests that brain has the capacity to synthesize, store and release ALLO into the extracellular fluid in concentrations that may be sufficient for a pharmacological action on neurons. In this project we will measure ALLO ant other neurosteroids in discrete brain areas and in brain microdialysates from normal and ADX/CX or hypophysectomized rats stimulating neurosteroid synthesis by administering to the rats MDR agonists, by increasing brain PREG content infusing PREG- SO4 or by blocking neurosteroid degradation (i.e. injecting blockers of 3alpha-HSOR). We also plan to study the contribution that glial cells and neurons make to the synthesis of neurosteroids. In view of the pharmacological potency of neurosteroids in the regulation of neuronal activity, these studies may ultimately help to establish the physiological relevance of specific neurosteroids and to devise pharmacological tools targeted to control alterations in neurosteroid production associated with CNS disorders of mood, affect, learning and memory.